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From 2011.igem.org

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Contents 1 Colombia @ iGem Notebook 1.1 June 1.1.1 June 30: 1.2 July 1.2.1 July 1 1.2.2 July 5 1.2.3 July 6 1.2.4 July 7 1.2.5 July 8 1.2.6 July 11 1.2.7 July 12 1.2.8 July 14 1.2.9 July 15 1.2.10 July 19 1.2.11 July 21 1.2.12 July 22 1.2.13 July 27 1.2.14 July 30 1.3 August 1.3.1 August 3 1.3.2 August 5 1.3.3 August 6 1.3.4 August 9 1.3.5 August 11

Colombia @ iGem Notebook

Here you can find our daily work in the Lab!

June

June 30:

• Biobricks 1, 2, 3, 4 and 5 were resuspended
• Miniprep Solutions (I, II and III) were prepared.

July

July 1

• Biobricks 1, 2, 3, 4 and 5 were resuspended.

July 5

• Biobrick 2 presented no colonies.
• Colonies from bricks 1, 3, 4 and 5 were stinged.

Task:

To make LB medium (15x25mL)

July 6

• Biobricks 1, 3 and 4 were twice plated.
• Brick 5 didn't grow up.
• We've added Ampiciline (3.75mL) and Kanamicine (1.875mL) to the boxed from the previous day.
• LB medium was prepared (15x25mL).
• Liquid LB was prepared (400mL).

Task:

To add ampiciline and tetracicline to the boxes.

Claim the liquid LB

Scrape ....

Sting biobricks 2 and 5 (no clons)

Pick up the tubes with Merceditas

July 7

• Clons 2 and 5 didn't work out.
• Clons 1, 3 and 4 were planted in LB liquid.
• E. Coli inoculation in Coffee
• Kanamicine resistence plasmid: 0.2 optic density.
• Direct inoculation x 2 and C(-) MgCl2.

Task:

Print electroporation protocol.

Ask Juan D. Olarte about the inoculation in coffee.

Minipreps for confirmation of 1, 3 and 4.

Competent cells for clons 2 and 5.

July 8

• Minipreps for 1, 3 and 4 were made.
• Plant sheets of coffee into different plates: Each sheet was cut in the middle and they were incubated at 25°C and 37°C LB+Kan.

Task:

To finish the minipreps from the addition of RNAsa.

Check the E. Coli growth in coffee.

July 11

• Minipreps have been finished. They've been planted in gel: Low concentration (25 ng/uL).
• Transformation protocol in chemical cells.

Task:

Prepare 250 mL of SOC medium

Print the Transformation protocol for chemical cells.

July 12

• Strains 1 and 4 have been conserved.
• LB liquid culture was prepared again for brick 3.
• E. Coli growth results.

Task:

Print the Transformation protocol for chemical cells.

Competent cells for clons 2 and 5.

Preserve brick 3.

Confirm bricks 1, 3 and 4 (Digestion)

Resuspend all Biobricks.

Check the E. Coli growth in coffee.

July 14

• E. Coli didn't grow up on the sheets.
• Chemical competent cells were made (DH5α).
• Brick 3 was left to grow in liquid medium.
• Bricks 2, 5, 6,7, 8, 9 and 10 were transformed and plated.

Task:

Print the Transformation protocol for chemical cells.

Preserve brick 3.

Confirm all biobricks (Digestion)

Sting the transformed bricks.

Add antibiotic to the mediums made today.

July 15

• Brick 3 was preserved in Revco.
• Bricks 2, 5 and 8 were stinged.
• 25 LB+Kan boxes x 25 mL.
• No colonies in brick 6.
• Bricks 7, 9 and 10 were contaminated.

Task:

Print the chemical cells protocol.

Confirm all biobricks (Digestion).

July 19

• Pass strain of Vibrio fischeri to blood agar base.
• Check the growth of the isolates
• Pass the transformed bricks 6, 7, 9 and 10.
• Pass the isolates the solid media to liquid media (2,4,5 and 8)

July 21

• Digestion to confirm No. 1, 3 and 4

Reactives	1X	6X
H2O	29,4µL	160,4 µL
Buffer N. 3	4 µL	24 µL
EcoRI	0,3 µL	1,8 µL
PstI	0,3 µL	1,8 µL
DNA	7 µL	40 µL

July 22

• Minipreps
• Electrophoresis of the digestions

Task:

Confirm minipreps

Re-suspend primers

July 27

• To prepare LB and SOC medium and autoclaved
• The bricks 7, 9 and 10 were again transformed and plated
• Plate the brick 6.

July 30

• Minipreps with RNase.
• Agarose gel Electrophoresis—Results were not obtained. REPEAT!!

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August

August 3

• PCR 16S to DNA Vibrio fischeri U. Nacional
• To expected a band of 1400 bp.

Reactives	1X	3X
H2O	6,8µL	20,4µL
Bµffer	1µL	3µL
MgCl2	0,8µL	2,4µL
dNTPs	0,2µL	0,6µL
Fw7	0,2µL	0,6µL
Rv49	0,2µL	0,6µL
Taq	0,1µL	0,3µL
DNA	1µL	-
	10µL

PCR Conditions

94 C for 5 min

95 C for 50 sec

55 C for 45 sec 35X

72 C for 1:30 min

72 C for 12 min

12 C forever

August 5

• Re-suspend primers

100µM → 10 µM

Example: igem 1 → 36.1 nm → 361 µL H2O igem 2 → 32.2 nm → 322 µL H2O ➱ Vortex

Vf= 50 µL Ci= 100 µL Cf= 10 µL Vi= ?

Vi= 5 µL

• Diluted DNA Vibrio fischeri

1244.1 ng/ µL

Ci= 1244.1 ng/ µL

Cf= 25 ng/ µL

Vf= 50 µL

Vi= 1 µL + 49 µL H2O

August 6

• Solutions 2 and 3 of miniprep
• Transformations of 2, 5, 7 and 13
• Check primers

Inventory

The petri dishes with bacteria are in Q401

PCR genes (sensor, CBP and chitoporin) of Vibrio fischeri with Pfx

Name	Dir	Gene	TM
Igem 1	Fw	sensor	48,8 C
Igem 2	Rv	sensor	50,8 C
Igem 3	Fw	CBP	48,9 C
Igem 4	Rv	CBP	49,1 C
Igem 5	Fw	Chitopor	49,3 C
Igem 6	Rv	Chitopor	49,4 C

August 9

• Minipreps:

1	✔
2	✕
3	so-so
4	✔
5	✕
8	so-so
9	so-so
10	so-so

August 11

• PCR chiA of Vibrio fischeri with Pfx

Name	Dir	Gene	Tm	Lenght
Igem 7	Fw	ChiA	55,4 C
Igem 8	Rv	ChiA	53,3 C	3378 pb